Method of purifying acidic components of a hop extract

ABSTRACT

A method for separating isohumulones from lupulones and residual hop constituents as contained in e.g., an isomerized hop extract, by means of a strongly basic ion exchange resin.

United States Patent [191 Herwig et al.

[ 1 Sept. 18, 1973 [75] Inventors: William C. Herwig, Milwaukee;

Donald H. Westermann, Brookfield, both of Wis.

[73] Assignee: Miller Brewing Company,

Milwaukee, Wis.

[22] Filed: Sept. 22, 1967 [21] Appl. No.: 669,947

[52] US. Cl. 260/586 R, 99/505 [51] Int. Cl. C07c 45/24 [58] Field ofSearch 260/586, 586 R; 99/505 [56] References Cited UNITED STATESPATENTS 3,433,642 3/1969 Nakayama et al. 99/505 OTHER PUBLICATIONSArmononds et al. .Iourn. of the Institute of Brewing,

Vol. 6 p 495403 (1962).

Howard et a1. Jour. Inst. Brew. Vol. 66, pp. 305-312, 1960.

Alderton et a1., Analytical Chemistry Vol. 26 pp 983-992 (1954).

Simmonds et a1. Jour. Inst. Brew. Vol. 68 pg 495 (1962).

Howard et al. Jour. Inst. Brew. Vol. 66 pg 305 (1960).

Primary Examiner-Leon Zitvcr Assistant Examiner-Norman MorgensternAttorneyCharles L. Harness and Kenneth E. Prince [57] ABSTRACT A methodfor separating isohumulones from Iupulones and residual hop constituentsas contained in e.g., an isomerized hop extract, by means of a stronglybasic ion exchange resin.

6 Claims, 1 Drawing Figure METHOD OF PURIFYING ACIDIC COMPONENTS OF AHOP EXTRACT BACKGROUND OF THE INVENTION 1. Field of the Invention Hopshave been used traditionally in the brewing of beer and ales for thepast several centuries, imparting to the beverage a pleasing bitterness,mild hop aroma, and a creamy head of foam. The composition of hops isquite complex, consisting of not only cellulosic material and moisture,but nitrogenous matter, pectins, tannins, resins, essential oils andask. The most important components are the essential oils and theresins, which contribute the attributes of the hop.

The bitterness of beer is due primarily to the total soft resinfractions of hops, and, more specifically, to the alpha-acid content,providing that fresh hops are used. The most effective way to utilizehops in the brewing process is through addition of hop extracts; morespecifically, to add the desirable components of an extract at variousstages of the brewing process to produce required results. Thespecificity of organic solvents to selectively dissolve certain hopcomponentsis well known, dating back to the turn of the century. Thisphenomenon enabled hop chemists to separate out waxes, hard resins,total soft resins, tannins and pectins by proper selection of solvents.(For example, all comhumulone, lupulone and unidentified soft resinthrough the use of a silica gel column. Rigby and Bethune.

ponents except tannins and pectins are soluble in diethyl ether; waxesare insoluble in cold methanol; total soft resins are soluble in lightpetroleum hydrocarbons.) The property of preferential solubility is usedin the preparation of commercial extracts.

Solvent extracts of hops have been found to be stable for several yearseven when stored under adverse conditions, but most commerical extractsavailable on the market today must be added to the brew kettle in orderto convert the alpha-acids, humulones, to the more water soluble bitterisohumulones. However, when such extracts are used, only 25-33% of thepotentially available isohumulone'is found in the finished beer. Theremaining isohumulone precipitates with the wort proteins during boilingand cooling, precipitates from beer due to the drop in pH duringfermentation, is oxidized in the kettle boil, or is adsorbed on theyeast. lnsignificant amounts are lost through further processing of thefinished beer.

The commercial processes for preparing isomerized hop extract for beercommonly result in a mixture of isohumulones, lupulones, essential oils,soft resins, and sundry minor components. Of this mixture only theisohumulones are of real value in beer. The other materials contributelittle to flavor or the character of the foam.

Various methods of separating isohumulones from the aforesaid othercomponents of an isomerized hop extract are known. In general, however,these methods are costly and tedious, involving a number of solventextraction steps and generally not resulting a substantially 100 percentyield of isohumulones substantially free from the other hop componentsin the starting extract. The instant invention, on the other hand, doesin fact accomplish these desired aims while being economic andefficient.

2. Description of the Prior Art Some of the prior art methods in thisfield include the following. Beyaert and Cornard Chem. Abs, 42, 7927B(1948), were able to resolve the total soft resins into A.S.B.C. Proc.,1952, 98, separated humulone and lupulone from the other components ofthe total soft resin using countercurrent distribution and, with furthermodifications, were able to separate the isomerized alpha-acids intoisoadhumulone, isohumulone, and isocohumulone. Rigby and Bethune,A.S.B.C. Proc. 1953, l 19, also separated the alpha-acids into theiranalogs and applied these techniques to continuous countercurrentextraction of a hop extract, Can. U.S'. Pat. 619,563, and claimseparation into oils, soft resins, lupulones, hard resins and humulones.The latter were resolved into their analogs and subsequently isomerized.Alderton et al, Anal. Chem., 26,983 (1954), reported the adsorption ofhumulone and lupulone by a strongly basic ion exchange resin, such asAmberlite IRA 400, from alcoholic media but not from petroleum ether.Verzele and Govaert, Wall. Comm., 18,181 1955), were able to separatethe hop alpha-acids into their analogs using partition chromatography onbuffered silica gel with iso-octane as the moving immiscible phase.Spetsiz and Steninger, J. lnst. Brew., 62,333 (1956), were able toresolve the isohumulones in beer into their analogs with reversed phasepartition chromatography using silinized diatomaceous earth impregnatedwith carbon tetrachloride and aqueous buffered methanol as the movingimmiscible phase. Howard and Slater, .l. Inst, Brew., 66,305 (1960),were able to adsorb the acidic components of hops from a methanolicsolution on a basic ion exchange resin (Dowey 1 X 4) in the acetate formand eluted the components with a methanolic acetic acid gradient. Theacidic components could be separated from the remainder of the hopcomponents by eluting with aqueous methanolic sodium chloride. Simmondsand Wilson, J. lnst. Brew., 68,495 (1962), where able to separateisohumulone, isocohumulone, lupulone, colupulone, humulone, andcohumulone from one another using a basic ion exchange (Dowex 1 X 4)column in the acetate form with 20% 2M aqueous sodium acetate inmethanol at 40C. A preisomerized extract was also separated into itscomponents using a similar system. In the two previously described ionexchange systems the resin and the extract are in a polar, water solublevolvent (methanol) system.

SUMMARY OF THE INVENTION In summary, this invention is directed toseparating the isohumulones from lupulones and residual hop constituentsin an isomerized hop extract dissolved in a water-immiscible organicsolvent by the process comprising bringing the solution into intimatecontact with particles of a strongly basic ion exchange resin containingan aqueous solution of a water-miscible solvent (preferably but notnecessarily a lower alkanol), the interstices between the particlesbeing occupied by a waterimmiscible organic solvent, whereby saidisohumulones are selectively taken up or adsorbed by the resin, followedby eluting the said isohumulones from the resin with a lower-alkanolicaqueous solution of a salt to give a final solution of said isohumulonessubstantially free from said lupulones and residual hop constituents.

The process of the invention is shown in diagrammatic form in thedrawing. The process of the invention may be described generally asfollows. The basic anion exchange resin must first be put in suitableform to separate isohumulates from lupulones. These resins are generallyavailable in chloride form, and if obtained in this form must beconverted to the form of a salt of a weak acid, suitably to the acetateform. This is done by placing the resin in the column and washing withan aqueous solution containing ions of a weak acid, e.g., with aqueousacetic acid, sodium acetate solution (1.0 N) or the like. The resin iswashed free of excess acetate or like ions with water. The resin, now inthe form of a salt of a weak acid, e.g., in the acetate form, is washedwith a water-miscible solvent (e.g., 50-90 percent vol. percentmethanol) to allow inclusion of the said solvent into the interiors ofthe resin beads or particles.

The step next to be described is optional. If desired, it can be omitted(cf. Example 1 with only a very slight reduction in yield. In thisoptional step the watermiscible solvent present in the intersticesbetween the resin particles is washed out with an inert waterimmiscibleorganic liquid solvent for isohumulones, e.g., hexane. This treatmentdoes not operate to remove the water-miscible solvent or liquid, e.g.,aqueous alkanol, from within the resin beads, but only from the spacebetween the beads, i.e., from their interstices. This water-immisciblesolvent is preferably saturated with the aforsaid water-miscible solventbefore use in this step. For example, if hexane is used as thewaterimmiscible solvent in this step, and if methanol-water is used asthe water-miscible solvent, the hexane is preferably saturated with themethanol-water solution, to reduce extraction of aqueous phase from theinteriors of the particles into the nonaqueous phase in the resininterstices. The system at this point (using this optional step)therefore consists of resin beads swollen with water-miscible liquid,with their interstices filled with the water-immiscible organic liquid(e.g., hexane saturated aqueous alcohol). If this optional step is notused, the interstices between the beads will of course still be filledwith the water-miscible solvent or liquid. In either case, the resin bedis now ready for use in the extraction process next described.

An extract of hops (isomerized, or isomerized and reduced), and alsocontaining lupulones and residual hop constituents, in an inertwater-immiscible organic liquid solvent, and which can (though it neednot be) the same as the inert water-immiscible organic liquid referredto in the preceding paragraph, is added to the column. In this step theresin bed retains both he isohumulate anions and the hydrogen ions fromthe hop acid (isohumulones), whereas the lupulones and residual hopconstituents pass through with the effluent. The acetate or like anionsin the resin particles are not exchanged to the effluent stream, butrather are apparently exchanged within the resin particles, and at thisstage stay with the particles.

Next, water-miscible solvent, e.g., 50-90 vol. percent methanol ispassed through the resin bed to elute the acid hydrogen (hydrogen ions)along with acetate ion, as acetaic acid. Minor amounts of residuallupulone and degradation materials, etc. are also washed out in thisstep.

Next, a solution ofa salt the anion of which is capable of displacingthe isohumulate anion, viz., an alkali metal salt, e.g., 1-5 g./l ml. ofsodium acetate in aqueous alkanol 1.17g/l00 mm. of sodium chloride inaqueous methanol (Example 2); 5.12g/100ml of ammonium acetate in aqueousmethanol (Example 8);

3.84g/l00ml of sodium propionate in aqueous methanol (Example 12); or2.62g/l00ml of sodium formate in aqueous methanol (Example 13), ispassed through the resin bed. This replaces the isohumulate anions withanions of the alkali metal salt, e.g., with acetate anions, withsimultaneous regeneration of the resin in such case to the acetate formand elution of the replaced isohumulate ions. The effluent, or eluate,consisting essentially of alkali metal (e.g., sodium) isohumulates inaqueous alkanol, with some sodium acetate (when this salt is used), andsubstantially free from lupulones and residual hop components, isrecovered and may be treated further to cover pure isohumulates byconventional procedures.

The regenerated resin bed is now ready for further use, and theextraction process can be repeated, optionally starting with the washwith hexane or the like above described, to replace the aqueous alkanolin the interstices between the resin particles or starting with thedirect addition of hop extract as in Example 1.

It has been mentioned that the basic ion exchange resin is preferablyused in acetate form. Actually, other forms can be used, in which theanion is that of a fairly weak acid, i.e., an acid substantially nostronger than formic acid.

The water-miscible solvent or liquid used throughout is preferably alower alkanol, e.g., methanol, ethanol, isopropanol, or the like.However substantially any neutral, inert water-miscible liquid isoperable, e.g., acetone, dimethyl sulfoxide, and the like. As mentioned,these are used in aqueous solution. The amount of water-miscible liquidin the solution should be at least 50 vol. percent, and can be as highas A preferred range is 6780 percent. If desired, two or three differentsuch liquids can be used in the respective steps, e.g., aqueous methanolin the first inclusion step, aqueous ethanol in the elution of acidhydrogen, and aqueous isopropanol in the final isohumulone elution; orany permutation of water-miscible liquids can be used in these steps. Itis preferred, however, that the same liquid be used throughout for thesake of simplicity and ease of recovery and reuse.

When the optional varient is employed in which an inert water-immiscibleorganic liquid used to treat the resin bed to fill the intersticesbetween the resin particles, the nature of that liquid is not critical,the only requirement (in addition to water-immiscibility) being that itbe a solvent for isomerized hops. The same considerations apply to thesolvent for the initial hop extract. Numerous such liquids are known andavailable for both uses, especially hydrocarbons and chlorinatedhydrocarbons.

Although it is preferred, the solvent for the starting solution of hopconstituents and the solvent used to fill the interstices between theresin beads actually need not be identical. However, if two differentsolvents are used, they should be mutually miscible. For example, theresin interstices can be filled with benzene, and the hop solution canbe in cyclohexane, since these two hydrocarbons are mutually soluble inall proportions. And there are many other suitable pairs of differentsolvents, obvious to these skilled in the art.

It is immaterial which solvent is in the interstices of the resin beadand which is used as the solvent for the initial hop solution. Thehydrocarbons are preferred for both uses because they are cheaper. Thus,such liquids can be substantially any inert normally liquid hydrocarbon,e.g., the liquid alkanes and aromatic hydrocarbons, e.g., benzene,toluene, xylene, and the like; and mixtures of these, e.g., petroleumether. Suitable liquids other than hydrocarbons include: carbon.tetrachloride, trichlorethylene, methylene dichloride, and the like.

FURTHER DESCRIPTION OF THE INVENTION As used in the art, and in thisspecification, the term isohumulones refers to the group of humulonesresulting from isomerization of the alpha acid components of hops, andto their reduced forms. Thus the term includes isohumulone,isocohumulone, isoadhumulone, isoprehumulone, and isoposthumulone, aswell as the reduced forms of these compounds. Said reduced forms arewellknown and areobtained by reduction of the starting isohumulones withborohydrides, or hydrogen, etc. The isohumulones as suchare weak acids.The term conventionally (and herein) includes their acid as well astheir salt forms, as for example, in the case that they are neutralizedwith sodium hydroxide or other base. In salt or ionic form they, theisohumulones, are frequently referred to as isohumulates, e.g., sodiumisohumulate, etc.

Similarly, as used herein, the term lupulones and residual hopconstituents? means materials other than isohumulones, present inisomerized hop extract. As already mentioned; these materials comprisemostly the lupulones (including its reduced forms), essential oils, softresins, and sundry minor components. A typical analysis of an isomerizedhop extract made by a hydrocarbon solvent extraction follows and'showsthe relative typical ranges of these materials.

California Isomerized Extract Made Seedless Hops from Other VarietiesTypical Content Range lsohumulnes 40% l550% Lupuloncs 25% lO-25% SoftResins 25% Ca. 25% Waxes 5% Ca. 5% Oil 4% l-5% The strongly basic ionexchange resins required for use in this invention are well known, andtheir preparation and properties have been extensively described inliterature. See Nachod and Schubert, Ion Exchange Technology, AcademicPress (1956); Osborn, Synthetic Ion Exchangers, Chapman and Hall, Lts.(1955); Kunin, Elements of Ion Exchange,Reinhold (1960); US. Pat. No.2,59l,573; Calmon and Kressman, Ion Exchangers in Organic andBiochemistry, lnterscience Publishers, Inc. (1957). They arecommercially available under various trade marks, e.g., Dowex l, Dowex21K (Dow Chemical Co.), Amberlite IRA 400 (Rohm and Haas Co.), and thelike.

In a typical preparation of a strongly basic ion exchange resin, acrosslinked divinyl benzene copolymer resin is first made by suspensionpolymerization. It is then treated'with chloromethyl methyl ether in thepresence of a Friedel-Crafts catalyst to attach chloromethyl groups tothe benzene rings in the polymer. These chloromethyl groups are thentreated with a tertiary amine such as N(CH thereby giving thecorresponding quaternary ammonium chloride, -CH N (CH Cl. This gives aso-called type 1 resin. Or the chloromethyl group can be quatemarizedwith an ethanolamine to give the so-called type 2 resin. It is availablecommercially in this chloride form. These materials are so stronglybasic that they are difficult to convert from the commercially availablechloride form to the free base form, even with caustic aoda. For use inthe instant invention, the resin should be converted to a weak anionform, preferably to the acetate form, as hereinafter described.

Strongly basic ion exchange resins are conventionally available inlightly-to-medium crosslinked forms, with about 2-4 percent crosslinking(e.g., with divinyl benzene), and these are preferred for use in thisinvention. More highly cross-linked materials, e.g., 8 percent and up,require increased adsorption and elution cycles, with reduction inyield.

Although we do not wish to be bound to any particular theory as to howthe process of this invention operates, our experience with it indicatesthe following mechanism (exemplified with methanol as the watermiscibleliquid, hexane as the water-immiscible liquid, and using the resin inacetate form. When the isomerized hop extract in hexane contacts theaqueous methanol-swollen ion exchange resin, the dissolved isohumulateions displace acetate ions within the resin. At this stage the resinalso acts partly as a screen, in that the lupulones and residual hopconstituents, even though dissolved in the incoming hexane solution, arenot absorbed in the resin pores and in large part pass completelythrough the column. Any residue is removed from the column by asubsequent hexane wash. This now leaves the desired isohumulate ionsadsorbed in the resin pores, said pores still being swollen with aqueousmethanol.

The next step operates to remove the interstitial hexam from the resinbed by washing with aqueous methanol. During this aqueous methanol wash,acetate ion which had been exchanged by isohumulate ion and weaklyacidic hop components are eluted from the column. The resin stillcontains isohumulate ion which can be removed by other ions, e.g.,chloride or the like. To do this, the resin is eluted with, e.g.,methanol-sodium acetate solution. The acetate ions replace theisohumulate ions in the resin pores, and the latter are washed out withthe elution liquor, thereby to give an aqueous alcohol solution of.sodium isohumulate which contains also some sodium acetate, but issubstantially free from lupulones and residual hop constituents as wellas hexane. This solution is now in a form that can be added directly tobeer or, if desired, the isohumulones can be recovered therefrom byconventional means.

For example, the solution can be acidified and extracted with petroleumether. This takes up the isohumulones and leaves the salt behind in theaqueous phase. The petroleum ether solution of isohumulones is thenrecovered and the solvent is evaporated off, leaving substantially pureisohumulones as residue.

Thus, we have discovered a new method of separating the acidiccomponents of hops, especially from a tion. A process of theseattributes has heretofore not been accomplished.

More particularly, we have discovered that the acetate form of astrongly basic crosslinked ion-exchange resin swollen with aqueousmethanol will quantitatively remove isohumulone or reduced isohumulone(e.g., catalytically hydrogenated isohumulone or borohydride reducedisohumulone) from a hexane extract of hops. The adsorbed material can bequantitatively recovered from the column by eluting e.g., withmethanolic aqueous sodium acetate solutions. The column is prepared andoperated by the general procedure described below.

Approximately 50 ml. of wet, strongly basic ion exchange resin, 4percent cross-linked (Dowex l X 4, 20-50 mesh) in the commerciallyavailable chloride form was slurried with water overnight. The resin waspoured into a 1.1 X 60 cm. column and washed with lN sodium acetatesolution until the effluent from the column was free of chloride ions.The column was washed free of sodium acetate with deionized waterfollowed by aqueous methanol. (Subsequent examples will show thepreferred range of alcohol concentration in the aqueous methanolsystem.) The interstitial aqueous methanol phase was replaced withhexane by descendingly washing out the heavier phase with hexanesaturated with aqueous methanol. (The step immediately preceding isoptional; it can be omitted, but there will often be a small reductionin yield.) A hexane solution of hop extract containing isohumulone orreduced isohumulone was passed down through the column with hexane,whereby isohumulones in the extract were adsorbed by the ion exchangeresin. The interstitial hexane was flushed from the column ascendinglywith aqueous methanol. The adsorbed hop acids, i.e., isohumulones, wereeluted from the column ascendingly with 4:l methanol-2N sodium acetatesolution. The eluate from the column contained substantially all thepure isohumulones of the original extract, substantially free fromlupulones and residual hop constituents. the acidic components wererecovered from the eluate by evaporating off the water and methanol, oralternately by extracting the isohumulone by conventional methods wellknown to those skilled in the chemistry of hop compounds.

Instead of methanol, other lower alkanols can be used, e.g., ethanol,isopropanol, and the like.

Any water soluble compound with an anion with greater affinity for theion exchange resin than the isohumulate anion will serve to displace theisohumulate anion, for example, chlorides, hydroxides, acetates,formates, and the like.

Of the foregoing, sodium acetate, potassium acetate and ammonium,acetate, as well as the corresponding chlorides, are particularlypreferred in the elution of isohumulone, catalytically hydrogenatedisohumulone or sodium borohydride reduced isohumulone. The elution maybe run ascendingly or descendingly at room temperature or elevatedtemperatures prudent to good practice. The aqueous alcohol phase in theresin preferably contains at least 50 volume alcohol, with at least 5volume percent water. Experience has shown that, within these ranges,column capacity increases as the alcohol concentration increases.

It will be immediately evident to those skilled in the art that numerousmodifications are possible within the description of the inventionherein given. For example,

I the solvent for the starting solution of hop constituents may be anyorganic solvent immiscible with the solvent system used to swell theresin.

The solvent system used to swell the resin must consist of water plus aninert water-miscible organic solvent.

The following examples illustrate without limiting the invention.

EXAMPLE 1 Approximately 50 ml. of Dowex l X 4 (20-50 mesh), a stronglybasic lightly cross-linked ion-exchange resin in chloride form wasslurried with water and poured into a L! X 60 cm. column and convertedto the acetate form by passing 250 ml. of N sodium acetate solutionthrough the column. The column was then washed with 100 ml. of 3:1methanol-water, and 20 ml. of a hexane solution of isomerized and sodiumborohydride reduced hop extract prepared by the general procedure ofExample 5 of U. S. Pat. No. 3,044,879 and containing 2,876 g. of reducedisohumulone was passed directly through the column followed by hexaneuntil the effluent was practically colorless (40 ml.).

The interstitial hexane was flushed from the column ascendingly with ml.of 3:1 methanol-water and the reduced isohumulone eluted from the columnwith 4:1 methanol-2M sodium acetate solution yielding 2.831 g. ofreduced isohumulone free from lupulones, etc., in 200 ml. of eluate.

EXAMPLE 2 Approximately 50 ml. of Dowel 21K resin (20-50 mesh), astrongly basic lightly crosslinked ionexchange resin in chloride formwas slurried with water and poured into a 1.1 X 60 cm. column andconverted to the acetate form by passing 250 ml. of N sodium acetatesolution through the column. The residual sodium acetate was washed fromthe column with ml. of deionized water followed by 100 ml. of 111methanol: water. The interstitial aqueous methanol phase was replacedwith hexane. 10 ml. of a hexane solution of an isomerixed and sodiumborohydride reduced whole hop extract, prepared by the general procedureof Example 5 of U. S. Pat. No. 3,044,879, and containing about 1.094 g.reduced isohumulone with 2.0 g. lupulones and residual hop constituentswas passed through the column. After washing out the interstitial hexanewith 2:1 methanolzwater, the adsorbed reduced isohumulone was elutedwith 4:1 methanol-N sodium chloride solution yielding L093 g. ofisohumulones free from lupulones, etc. in 250 ml. of eluate.

EXAMPLE 3 The column in Example 2 was regenerated and prepared for useas described in Example 1. 10 ml. of a hexane solution of sodiumborohydride reduced whole hop extract solution containing 1.084 g. ofreduced isohumulone and obtained as in Example 1 was passed through thecolumn, washed through the column with 40 ml. of hexane followed byascendingly washing the interstitial hexane out with 75 ml. of 2:1methanol:water. The adsorbed material was eluted off with 4:1 methanol:2N sodium acetate solution at 40C. yielding 0.981 g. of isohumulonessubstantially free from other hop constituents in 200 ml. of eluate.

EXAMPLE 4 The column in Example 3 was washed ascendingly with 100 ml. of1:1 methanohwater to remove any fines and the interstitial aqueous phasereplaced with hexane. m1. of a hexane solution of sodium borohydridereduced whole hop extract feed solution containing 1.169 g. of reducedisohumulone prepared as above was passed through the column and washedwith 40 ml. of hexane. The interstitial hexane was washed from thecolumn with 75 ml. of 2:1 methanolzwater and the adsorbed materialeluted ascendingly with 4:1 methanol- 2N sodium acetate solution at roomtemperature yielding 1.135 g. of reduced isohumulone substantially freefrom other hop constituents in 300 ml. of eluate.

EXAMPLE 5 EXAMPLE 6 The column from Example 5 was washed with 100 ml. of3:1 methanolzwater and the interstitial aqueous phase replaced withhexane. ml. of a hexane solution of sodium borohydride reduced whole hopextract feed solution prepared as above and containing 2.260 g. ofreduced isohumulone was passed through the column followed with 40 ml.of hexane. The hexane phase was replaced by washing ascendingly with 75ml. of 3:1 methanolzwater followed with 4:1 menthanol-2N sodium acetatesolution yielding 2.254 g. of recovered isohumulone material in 250 ml.of eluate, substantially free from lupulones, etc.

EXAMPLE 7 50 ml. of a hexane solution of sodium. borohydride reducedwhole hop extract feed solution prepared as above and containing 5.650g. of reduced isohumulone was passed through the column prepared as inExample 6 and washed with hexane until the effluent was colorless. Theinterstitial hexane was flushed from the column with 75 ml. of 3:1methanolzwater and the adsorbed material eluted ascendingly with 4:1methanol- 2N sodium acetate solution yielding 5.518 g. of isohumulonematerial in 300 ml. of eluate, substantially free from lupulones andresidual hop constituents.

EXAMPLE 8 10 ml. of a hexane solution of sodium borohydride reducedwhole hop extract feed solution prepared as above and containing 0.950g. of reduced isohumulone was passed through the column prepared as inExample 6 and washed through with 50 ml. of hexane. The interstitialhexane was flushed from the column with 75 ml. of 3:1 methanolzwater andthe adsorbed material eluted with 2:1 methanol-2N ammonium acetatesolution yielding 0.991 g. of reduced isohumulone, substantially freefrom lupulones and residual hop constituents.

EXAMPLE 9 The column in Example 5 using Dowex l X 4 (20-50 mesh) wasregienrated by previous elution of an extract using 4:1 methanol-2Nsodium acetate solution. The column was washed with 100 ml. of 3:1methanol-water and the interstitial aqueous phase replaced with hexanesaturated with aqueous phase solution (3:1 methanol:- water). 20 ml. ofa hexane solution of isomerized hop extract containing 2.28 g.isohumulones and 6.124 g. of total soft resin was passed through thecolumn followed by 30 ml. of hexane. The hexane in the interstices wasreplaced by washing ascendingly with 75 m1. of 3:1 methanolzwater. Theadsorbed isohumulone was eluted with 4:1 methanol-2N sodium acetatesolution yielding 2.013 g. of isohumulone in 250 ml. of eluatesubstantially free of lupulone and other hop components.

EXAMPLE 10 The column from Example 9 was washed with 100 ml. of 3:1acetone-water and the interstitial aqueous phase was replaced withhexane saturated with aqueous phase. Twenty ml. of a hexane solution ofan isomerized and sodium borohydride reduced hop extract containing2.818 g. of reduced isohumulone was washed through the column withhexane, recycling the first 10 ml. of effluent.

The interstitial hexane was flushed from the column ascendingly with 3:1acetone-water followed by 3:1 methanol-water. The reduced isohumulonewas eluted from the column with 4:1 methanol-2M sodium acetate solutionyielding 2.026 g. of reduced isohumulone in 250 ml. of eluate.

EXAMPLE 1 1 The column from Example 10 was washed with 100 ml. of 3:1dimethyl sulfoxide-water and the interstitial aqueous phase was replacedwith hexane saturated with aqueous phase. Twenty ml. of a hexanesolution of an isomerized and sodium borohydride reduced hop extractcontaining 2.920 g. of reduced isohumulone was washed through the columnwith hexane, recycling the first 10 ml. of effluent.

The interstitial hexane was flushed from the column ascendingly with 3:1methanol-water until 50 ml. of aqueous phase had passed through thecolumn. The reduced isohumulone was eluted .from the column with 4:1methanol-2M sodium acetate solution yielding 2.312 g. of reducedisohumulone in 200 ml. of eluate.

EXAMPLE 12 The column from Example 11; was converted to the propionateform of the resin by .washing with 250 ml. of N sodium hydroxidefollowed by ml. of deionized water and finally with 250 ml. of N sodiumpropionate solution. The column was washed free of sodium propionatewith deionized water and finally with 100 ml. of 3:1 methanol-water. Theinterstitial aqueous phase was replaced with hexane saturated withaqueous phase. Twenty ml. of a hexane solution of isomerized and sodiumborohydride reduced hop extract containing 2.974 g. of reducedisohumulone was passed through the column followed by 30 ml. of hexane,the first 10 ml. through the column was recycled through the column.

The interstitial hexane was flushed from the column ascendingly with 3:1methanol-water until 50 ml. of the aqueous phase had passed through thecolumn. The adsorbed reduced isohumulone was eluted with 4:1 methanol-2Msodium propionate yielding 2.875 g. in 200 ml. of eluate.

EXAMPLE 13 The column from Example 12 was converted to the formate formof the resins by washing with 250 ml. of N sodium formate solutionfollowed by 100 ml. of deionized water and then 100 ml. of 3:1methanol-water. The interstitial aqueous phase was replaced with hexanesaturated with aqueous phase. Twenty ml. of a hexane solution ofisomerized and sodium borohydride reduced hop extract containing 2.876g. of reduced isohumulone was passed through the column followed by 30ml. of hexane, the first ml. of efi'luent was recycled.

The interstitial hexane was flushed from the column with 3:1methanol-water until 50 ml. of the aqueous phase had passed through thecolumn. The adsorbed reduced isohumulone was eluted from the column with4:1 methanol-2M sodium formate yielding 2.216 g. in 200 ml. of eluate.

EXAMPLE 14 A column was prepared as described in Example 1 using Dowex 1X 4 (20-50 mesh). 10 ml. of a hexane solution containing 1.246 g. oftetrahydroisohumulone, prepared according to the method of Brown et al.,(J. Chem. Soc., 1959, 545), was passed down the column and washedthrough the column with hexane. The interstitial hexane was flushed fromthe column by ascendingly washing with 3:1 methanol-water until 50 ml.of aqueous phase has been collected. The adsorbed tetrahydroisohumulonewas eluted from the column ascendingly with 4:l methanol-2M sodiumacetate solution yielding 1.250 g. of material in 200 ml. of eluate.

EXAMPLE 15 EXAMPLE 16 The column from Example 14 was washed with 3:1

methanol-water and the interstitial aqueous phase re- I placed withtrichloroethylene. 7.25 ml. of a trichloroethylene solution containing0.648 g. of isohumulone was washed through the column with approximately30 ml. of trichloroethylene. The interstitial trichloroethylene wasreplaced descendingly with 3:1 methanolwater until 50 ml. of aqueousphase has passed through the column. The adsorbed isohumulone was eluteddescendingly from the column with 4:1 methanol-2M sodium acetatesolution yielding 0.634 g. of isohumulone in 240 ml. of eluate.

The isohumulones eluted as described above after concentrating orcrystallizing may be added to unhopped beer anytime during the brewingprocess resulting in substantial increase in hop utilization withretention of those attributes generally considered desirable in aconventionally hopped beer.

The invention claimed is: l

1. In the method of separating isohumulones from lupulones and residualhop constituents in solution in an inert water-immiscible organic liquidselected from the group consisting of carbon tetrachloride,trichlorethylene, methylene, dichloride, hexane, petroleum ether,cyclohexane, benzene, toluene, and xylene, the improvement comprising:

i. contacting a bed of particles of a strongly basic ion exchange resinpretreated with the anion of a weak acid selected from the groupconsisting of formate, acetate, and propionate; said particles beingswollen with an aqueous solution of an inert watermiscible liquid withsaid solution of isohumulones,

lupulones, and residual hop constituents; with water-miscible liquidbeing selected from the group consisting of methanol, ethanol,isopropanol, acetone, and dimethylsulfoxide; washing the resulting resinbed with 50-95 volume percent aqueous solution of an inertwater-miscible liquid selected from the group consisting of methanol,ethanol, isopropanol, acetone, and dimethylsulfoxide;

iii. eluting the resulting resin bed with a 50-95 volume percent aqueoussolution of an inert watermiscible liquid selected from the groupconsisting of methanol, ethanol, isopropanol, acetone, anddimethylsulfoxide, said solution containing l-5, l2g/l00 ml of anionized compound selected from the group consisting of ammonium acetate,sodium acetate, potassium acetate, sodium propionate, and sodiumformate; and

iv. recovering an eluate consisting essentially of a solution of saidisohumulones and said compound in said respective water-miscible liquid.

2. The method according to claim 1 in which the water-immiscible liquidis n-hexane.

3. The method according to claim 1 in which the water-miscible liquid isa methanol: water solution of 2-4 volumes of methanol per volume ofwater.

4. The method according to claim 1 in which the anion of a weak acid ini) is acetate.

5. The method according to claim 1 in which the ionized compound issodium acetate in l-5 g./ ml concentration.

6. The method according to claim 1 in which the isohumulones arepre-reduced.

UNITED STATES PATENT IIOFFICE v CERTIFICATE OF. CORRECTION Patent No.593 Dated Sept. 18. m3

Inventofls) ilfiillzlam-C. Her-wig and Donald H. Weltetmgnn It iscertified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown' below:

L3. m v To Read C01. 1, line 35 "comerical." v --commercia .1--

001. 2, line 3.1 "Eowcy" -..oowex cm. 4, Tine 43 "flue t" var a t Y C01.8, line 22 I "2,876" e v ---2,876-..

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I C01. 12. fines 40-41 T'l-SJZg/IOO" -1'--s.12 3/10 Sign ed and seale dthis 22nd day offiJelnuar'y 197M;

. SEAL) Attest:

EDWARD M. FLETCHERJRQ RENE D. TEGTMEYER Attesti ng Officer" ActingCommissioner "of Patents

2. The method according to claim 1 in which the water-immiscible liquidis n-hexane.
 3. The method according to claim 1 in which thewater-miscible liquid is a methanol: water solution of 2-4 volumes ofmethanol per volume of water.
 4. The method according to claim 1 inwhich the anion of a weak acid in i) is acetate.
 5. The method accordingto claim 1 in which the ionized compound is sodium acetate in 1-5 g./100ml concentration.
 6. The method according to claim 1 in which theisohumulones are pre-reduced.